<p>Root phenotyping technology is an essential component of modern agriculture and plant science research. Conducting root-related research in a non-destructive manner is crucial for studying plant roots without damaging the plants themselves and allowing for time-series studies. The research aims to validate the efficacy of MISIRoot, an innovative root phenotyping technology, through the implementation of two projects. The first project focuses on the early detection of western corn rootworm, one of the most devastating corn rootworm species in North America, particularly in midwestern corn-growing areas. The second project focuses on the assessment of the soybean nodulation process, which is crucial for nitrogen fixation by Rhizobia living in the nodules on the soybean roots. The current state-of-the-art methods for western corn rootworm and soybean nodulation assessments still require the whole plant to be dug up, which causes irreversible destruction to the plant itself. Although recently developed root phenotyping methods such as minirhizotron, CT, and MRI scanners offer unique advantages in observing plant roots, their potential for field applications is currently limited.</p> <p>Data collection for both projects was carried out using MISIRoot, a minimally invasive plant root phenotyping robot that works in situ within natural soil. The MISIRoot system mainly consists of an industrial-level robotic arm, a mini-size camera with a lighting set, a plant pot holding platform, and image processing software for root recognition and feature extraction. MISIRoot can take high-resolution color images of the roots in soil with minimal disturbance to the root and reconstruct the plant roots' three-dimensional (3D) structure at an accuracy of 0.1 mm.</p> <p>For the first project, the MISIRoot system successfully distinguished the corn plants inoculated with western corn rootworm larvae from the healthy plants before the shoot section of the corn plants revealed significant differences. For the second project, the MISIRoot system successfully demonstrated its ability to differentiate soybean plants with and without nodules.</p>

Studies were conducted in 1990 and 1991 to investigate two kinds of soil insects that attack field corn in Virginia. Corn rootworms, the most important of which is the western corn rootworm (WCR), feed on corn roots and have been sampled effectively using yellow sticky traps. The spatial distribution of weekly counts of WCR on yellow sticky traps was investigated over three consecutive weeks in July of each year using Pherocon A.M. traps. The traps were cut in half, placed in a seven by seven grid, and separated by an average distance of 18.3 m. Data were collected in a total of 24 corn fields. Dispersion indices showed that the trap counts of WCR were aggregated. Further analysis using geostatistics revealed that the trap counts were nonrandomly distributed at least half the time at all population densities; however, the presence and type of spatial pattern was density-independent. Pests of germinating corn seeds comprise a guild of insects including seedcorn maggots, wireworms, and white grubs. No one method has yet been developed to sample all of the insects in the guild. A new method, the baited wire trap (BWT), was evaluated in 47 fields in 1990 and 1991. No significant differences were found between the proportion of seeds damaged by pests of germinating seeds in the BWT and seeds planted by hand at conventional plant spacings. The proportion of damaged seeds in the BWT was significantly less than that in the commercially-planted fields; however, the proportion of damaged seeds in the BWT was significantly greater than the stand loss in these same fields. Linear regression revealed a significant relationship between the proportion of damaged seeds in the BWT and simulated plantings, and the conventionally planted fields. Because of low coefficients of determination (R²), the BWT could not be used to accurately predict damage by the germinating seed pest guild. However, the BWT detected damage by all three members of the pest guild and was found to be significantly faster to use than the bait station method. / Master of Science

Cry6Aa1, une nouvelle toxine produite par Bacillus thuringiensis (Bt), agit comme insecticide sur la chrysomèle du maïs (WCRW). Dans cette étude, on démontre que Cry6Aa1 est une toxine formeuse de pores (TFP) en bicouches lipidiques planes (BLP). Contrairement aux autres toxines de Bt étudiées jusqu’à présent, la formation de pores par Cry6Aa1 ne requiert pas de prétraitement par protéases et se produit à des doses de toxine deux à trois ordres de grandeur plus faibles que celles nécessaires pour les autres toxines de Bt dans les mêmes conditions. La formation de pores par la forme non traitée de Cry6Aa1 dépend du pH; les pores obtenus ont des conductances comprises entre 31 et 689 pS en conditions symétriques de 150 mM de KCl; ils sont cationiques avec un comportement cinétique complexe. Les propriétés biophysiques des pores ne changent pas lorsque la toxine est traitée avec le suc du mésenthéron de l’insecte (Cry6Aa1 WCR1). Par contre, un traitement à la trypsine (Cry6Aa1 TT) modifie la conductance et la sélectivité des pores à pH 5,5 (le pH physiologique de l’intestin de WCRW). La reconstitution en BLP de fraction de membrane native du mésenthéron de WCRW affecte les propriétés des pores formés par Cy6Aa1. Les déterminants moléculaires du mode d’action de cette nouvelle toxine formeuse de pores semblent donc différer de ceux décrits précédemment pour d’autres toxines de Bt. La structure atomique tridimensionnelle de Cry6Aa1 vient tout juste d’être élucidée. Elle montre que la toxine adopte une conformation riche en hélices α qui ressemble fortement à celle de la TFP ClyA produite par E. coli. En se fondant sur les données disponibles pour ClyA, on a étudié l’effet de divers changements dans les régions N et C terminales de Cry6Aa1 sur sa capacité de former des pores en BLP. / Cry6Aa1 is a new toxin produced by Bacillus thuringiensis (Bt), which displays insecticidal activity against the Western corn rootworm (WCRW). The present work demonstrates that Cry6Aa1 is a pore-forming toxin (PFT) in planar lipid bilayers (PLBs). Contrary to other Bt toxins tested so far, pore formation by Cry6Aa1 does not require protease pretreatment and takes place at doses that are two to three orders of magnitude lower than those required for other Bt toxins under similar conditions. Pore formation by Cry6Aa1 is pH-dependent; the conductances of the pores range between 31 and 689 pS under symmetrical 150 mM KCl conditions; they are cationic and display a complex kinetic behaviour. The treatment of the toxin with midgut juice (Cry6Aa1 WCR1) does not change the biophysical properties of the pores. However, the treatment with trypsin (Cry6Aa1 TT) affects their conductance and selectivity at pH 5.5 (the WCRW gut physiological pH). The incorporation in PLBs of native membrane material from WCRW midgut affects the behaviour of the Cry6Aa1 pores. The molecular determinants of the mode of action of this new PFT appear therefore to differ from those reported before for other Bt toxins. The three-dimensional (3-D) atomic structure of Cry6Aa1 has just been elucidated. It shows that the toxin assumes an α-helix-rich configuration, which is quite similar to that of the ClyA PFT produced by E. coli. Based on the data available for ClyA, we have studied how different changes in the N- and C-terminal regions of Cry6Aa1 affect its pore formation ability in PLBs.

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Submitted by Eduardo Neves Costa null (costa_ne@yahoo.com.br) on 2016-08-09T18:18:35Z No. of bitstreams: 1 TESE - EDUARDO - 2016.pdf: 1669833 bytes, checksum: 57591de7cf7e037137c8ef2df91c4b70 (MD5) / Approved for entry into archive by Ana Paula Grisoto (grisotoana@reitoria.unesp.br) on 2016-08-12T13:14:35Z (GMT) No. of bitstreams: 1 costa_en_dr_jabo.pdf: 1669833 bytes, checksum: 57591de7cf7e037137c8ef2df91c4b70 (MD5) / Made available in DSpace on 2016-08-12T13:14:35Z (GMT). No. of bitstreams: 1 costa_en_dr_jabo.pdf: 1669833 bytes, checksum: 57591de7cf7e037137c8ef2df91c4b70 (MD5) Previous issue date: 2016-07-26 / Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) / O inseto Diabrotica speciosa (Germar) e o ácaro Oligonychus pratensis (Banks) representam uma das principais pragas da cultura do milho, no Brasil e nos EUA, respectivamente. Essas pragas são controladas principalmente via aplicação de inseticidas químicos; contudo, há alternativas para tornar o manejo dessas pragas menos impactante, como o uso de plantas resistentes. Assim, esse trabalho teve como objetivo avaliar: (1) a resistência de cultivares de milho crioulo a D. speciosa; (2) a resistência de híbridos de milho resistentes à seca ao ácaro O. pratensis; (3) os mecanismos relacionados com a resistência a essas pragas; e (4) definir, através de testes de metodologia de pesquisa, a melhor forma para a condução dos ensaios. Desse modo, o presente trabalho é constituído por quatro pesquisas. Na primeira pesquisa, foi avaliada a antibiose em 19 cultivares de milho crioulo a D. speciosa, investigando se a expressão dessa categoria de resistência estava associada com algum mecanismo morfológico (lignina, celulose, fibras, e hemicelulose) ou químico (fenóis totais). No segundo trabalho, estudou-se a tolerância em seis cultivares de milho crioulo, previamente selecionados no teste de antibiose. A tolerância foi correlacionada com alguns componentes que participam do metabolismo primário vegetal, como a clorofila, os carotenoides, a glicina betaína, e a prolina. Em um terceiro experimento, foi avaliada a oviposição de D. speciosa nas seis cultivares selecionadas, discutindo os resultados quanto ao comportamento do inseto. No quarto ensaio, estudou-se a antibiose e a tolerância a O. pratensis em híbridos de milho resistentes à seca. Nessa pesquisa, foi avaliado se marcadores moleculares associados com resistência a estresses bióticos amplificariam o DNA de plantas resistente e suscetível, e se seria visualizado polimorfismos nos padrões de banda do DNA quando examinados em gel de agarose. As cultivares de milho crioulo Pérola, Fortuna e Palha Roxa manifestaram antibiose a D. speciosa, sendo isso observado no desenvolvimento e na sobrevivência (Pérola e Fortuna), e na fertilidade (Palha Roxa) do inseto. Observou-se que as cultivares resistentes exibiram as menores perdas de lignina, celulose, fibras e hemicelulose nas raízes de milho injuriadas por larvas de D. speciosa. A cultivar Azteca, que foi a mais suscetível em teste de antibiose, se destacou como a mais tolerante ao ataque da praga, além de apresentar os maiores teores de clorofilas e carotenoides. No que diz respeito à preferência para oviposição, as fêmeas de D. speciosa ovipositaram igualmente em cultivares resistentes e suscetíveis. O ácaro O. pratensis encontrou dificuldades para se desenvolver e sobreviver no híbrido de milho resistente à seca Artesian. Seis genes relacionados com resistência a estresses bióticos foram encontrados nesse híbrido, enquanto que no híbrido Droughtgard, que foi suscetível ao ácaro, nenhum daqueles genes foi observado. Os resultados obtidos neste trabalho demonstram o potencial da resistência de plantas de milho para o controle de pragas agrícolas, fornecendo informações à respeito de alguns dos mecanismos envolvidos na resistência dos materiais estudados, contribuindo, assim, para o desenvolvimento do Manejo Integrado de Pragas / The insect Diabrotica speciosa (Germar) and the mite Oligonychus pratensis (Banks) represent one of the major maize crop pests, in Brazil and in the USA, respectively. These pests are controlled primarily through the application of chemical insecticides; however, there are alternatives to make the management of these pests less impactful, as the use of resistant plants. Thus, this work aimed: (1) to determine, through testing research methodologies, the best way to conduct the assays; to evaluate (2) the resistance of maize landraces to D. speciosa and (3) the resistance of drought resistant maize hybrids to the mite O. pratensis; and lastly, (4) to reveal the mechanisms associated with the resistance to these pests. Thereby, the present work is constituted by four researches. In the first research, the antibiosis to D. speciosa was assessed in 19 maize landraces, investigating whether the expression of this resistance category was related with some morphological (lignin, cellulose, fibers, and hemicellulose) or chemical (total phenols) mechanism. In the second work, the tolerance was studied in six maize landraces previously selected in the antibiosis test. The tolerance was correlated with some compounds which participate in the primary metabolism of plants, as the chlorophyll, the carotenoids, the glycine betaine, and the proline. In a third experiment, the oviposition of D. speciosa was assessed on the six cultivars selected, discussing the results regarding the insect behavior. In the fourth assay, the antibiosis and tolerance were assessed to O. pratensis in drought resistant maize hybrids. This research aimed also to evaluate whether molecular markers associated with resistance to biotic factors would amplify the DNA of resistant and susceptible plants, and if it would be visualized polymorphisms in the banding patterns of the DNA when examined in agarose gel. The maize landraces Pérola, Fortuna, and Palha Roxa manifested antibiosis to D. speciosa, and it was observed on the development and survival (Pérola and Fortuna), and on the fertility (Palha Roxa) of the insect. The resistant cultivars exhibited the lowest losses of lignin, cellulose, fibers, and hemicellulose in the injured maize roots by D. speciosa larvae. Interestingly, the cultivar Azteca, which was the most susceptible in the antibiosis test, stood out as the most tolerant to the pest attack, besides showing the highest amounts of chlorophyll and carotenoids. With regards to the oviposition preference, the D. speciosa females laid eggs equally on the resistant and susceptible cultivars. The mite O. pratensis found difficulties for the development and survival on the Artesian drought resistant maize hybrid. Six genes associated with resistance to biotic stresses were found in this hybrid, whereas in the susceptible hybrid, Droughtgard, no one gene was noted. The results obtained in this work displays the potential of the maize plant resistance for the control of agricultural pests, providing information as to some mechanisms involved in the resistance of the materials studied, contributing, thus, for the development of the Integrated Pest Management / CNPq: 140589/2013-9 / CNPq: 140744/2016-9 / CAPES PDSE: BEX 5710/14-6

Master of Science / Department of Entomology / Larry L. Buschman, Lawrent Buschman / This study evaluated whether using a cover crop with corn would increase the threat from spider mites in western Kansas because cover crops may serve as a winter host. This study also evaluated whether a cover crop could affect corn rootworm and other ground dwelling arthropods in the cornfield. In the first study, downy brome, Bromus tectorum L., was used as the winter cover crop. There were two trials repeated for three years each. Each trial included: two amounts of irrigation, downy brome, and herbicide to control weeds. In the first trial there were no significant differences in corn rootworm, Diabrotica virgifera LeConte, damage across treatments, because there were no differences in brome residue across the treatments. In the second trial, corn rootworm damage was significantly more in plots with higher amounts of downy brome residue. There were no differences in numbers of spider mites: Banks grass mites, Oligonychus pratensis (Banks) or twospotted spider mites, Tetranychus urticae Koch, across treatments. Spider mite populations appeared to be suppressed by the predatory mite Neoseiulus spp., which also overwintered in the cover crop. Corn rootworm samples taken from a no-till irrigation experiment were variable among irrigation treatments but indicated a trend for rootworm damage to increase with increasing irrigation. In the second study, winter wheat, Triticum aestivum L., was used as the winter cover crop. There were three trials repeated for three years each. Each trial included two amounts of irrigation and winter wheat and three amounts of herbicide to control weeds. Upon completion of the agronomy trials, the plots were split into two subplots and one was tilled. Pitfall traps were installed to capture ground dwelling arthropods: (Coleoptera: Carabidae), wolf spiders (Araneae: Lycosidae) and crickets (Orthoptera: Gryllidae). Four carabid genera were more common under no-till conditions. One was more common in tilled plots. Five carabid genera were more common in plots with a history of high weed densities. Two carabid genera were more numerous in plots with the history of a cover crop. Crickets were more common under no-till conditions. Wolf spiders were more common in no tillage with a history of a cover crop.

A decision support system for preventive integrated pest management (IPM) and nonpoint source (NPS) pollution control was designed, implemented and evaluated. The objective of the system was to generate plans at the farm level to satisfy economic and production goals while limiting risks of insect pest outbreaks, nitrate and pesticide leaching and runoff, and soil erosion. The system is composed of a constraint satisfaction planner (CROPS-LT), a modified version of CROPS (Stone, 1995), a farm-level resource management system (FLAME), an NPS module, which includes a weather generator, CLIGEN (Nicks et al. 1995), and an NPS distributed-parameter model, ANSWERS (Bouraoui, 1994), databases, a database engine and utility programs. The performance of the system was analyzed and performance enhancing features were added to increase the planner's ability to find near-optimal plans within a limited planning time. Using heuristics to sort potential crop rotations based on profit generally improved the planner's performance, as did removal of fields that were not suitable for growing target crops. Not surprisingly, the planner was best able to find plans for crops that can be grown in a variety of rotational systems. Throughout, the ability to apply environmental constraints selectively to individual fields greatly improved the planner's ability to find acceptable plans. Preventive IPM (PIPM) heuristics to control corn rootworms CRW (<I>Diabrotica virgifera virgifera</I> and <I>D. barberi</I>) were added to the planner. The model was represented and solved as a constraint satisfaction problem. Results indicated that plans obtained using PIPM heuristics had less risk of CRW damage, reduced chemical control costs, higher profit and reduced soil erosion as compared to a control plan. Linking the planner to the NPS model in a feedback control loop improved the planner's ability to reduce soil losses while preserving economic and production goals. / Ph. D.

Made available in DSpace on 2017-07-25T19:29:56Z (GMT). No. of bitstreams: 1 Paulo Gallo.pdf: 1634093 bytes, checksum: f84f8b79e7d1355234216b134966f01a (MD5) Previous issue date: 2012-11-13 / The presence of Diabrotica speciosa larvae causes damage to corn root system and reduction in grain yield, mainly in second season crops. Applying insecticide in the seed furrow can be effective, but this assumes a form of preventive control and implies higher production costs and environmental impact. We evaluated the efficacy of genotypes AG7000RW and DKB330RW (Cry3Bb1) and their isolines to reduce the damage caused by D. speciosa larvae to corn root system, the efficacy of the insecticide fipronil applied in the furrow, and the effect of the interaction of both treatments on the yield of maize hybrids, as well as the planting date when the most severe damage occurs. The efficacy experiment was carried out in Itaberá, SP, in no-till planting system after oat crop. The damage to the roots was evaluated 52 days after emergence, using a visual score according to its intensity. Stalk lodging was determined by counting the plants leaning more than 25º in relation to its own axis at harvest. Genotypes AG7000 and DKB330 with no insecticide application presented damage and the resulting scores were 1.62 and 1.85, whereas the scores of their Bt isolines were 0.25 and 0.24, respectively. Genotypes AG7000 and AG700RW had the highest percentage of matter and amount of dry root in relation to DKB330 and DKB330RW, regardless of insecticide application. Stalk lodging of 46.7% found in non-Bt genotype DKB330 with no insecticide application reflected negatively on the yield of this treatment. In contrast, with insecticide application, stalk lodging ranged from 0.2% to 2.2% between genotypes, not influencing their yield. Cry3Bb1 protein was effective in reducing the damage to the root regardless of the genotype tested. Genotypes with high yield potential and lower root mass may present higher yield response when protected by the toxin or insecticide in the presence of a large larval population. In the 2010/2011 season, we assessed the damage in non-Bt corn root at four planting dates in Itaberá (10/14, 11/3, 11/25, and 12/20) and three in Castro, PR (9/29, 10/21, and 11/11). In the following season, 2011/2012, we evaluated six planting dates in Itaberá (9/21, 10/18, 11/21, 12/28, 1/30, and 2/20) and four in Castro (9/20, 10/10, 10/30, and 11/20). We observed that, from the second planting date on, the root damage reached scores over 3.0 and remained above 4.0 in all other planting dates in Itaberá in both seasons. In Castro, only the seeding performed in 11/20/2011 reached a score above 3.0. The use of protection in corn root, by applying insecticide or toxin, presented a greater response in sowing carried out from October on in the region of Itaberá. / A presença de larva de Diabrotica speciosa danifica o sistema radicular do milho e causa redução na produtividade de grãos, principalmente nas lavouras de segunda safra. A aplicação de inseticida no sulco de semeadura pode ser eficaz, porém, esta forma de controle assume caráter preventivo e implica maior custo de produção e impacto ambiental. Avaliaram-se a eficácia dos genótipos AG7000RW e DKB330RW (Cry3Bb1) e de suas isolíneas na redução do dano da larva de D. speciosa na raiz do milho, a eficácia do inseticida fipronil aplicado no sulco de semeadura e o efeito da interação de ambos os tratamentos sobre a produtividade dos genóti os de milho, bem como a época de semeadura em que ocorre maior dano da larva. O experimento de eficácia foi instalado em Itaberá, SP, na safra 2011/2012, sob sistema de plantio direto na palha após cultura de aveia. Os danos nas raízes foram avaliados aos 52 dias após a emergência, atribuindo-se nota visual de acordo com sua intensidade. O acamamento foi determinado contando-se as plantas com inclinação maior que 25º em relação ao próprio eixo no momento da colheita. Os genótipos AG7000 e DKB330 sem inseticida apresentaram dano, e as notas resultantes foram 1,62 e 1,85, enquanto as notas de suas isolíneas Bt foram 0,25 e 0,24, respectivamente. Os genótipos AG7000 e AG700RW obtiveram os maiores percentuais de matéria e quantidade de massa seca na raiz em relação a DKB330 e DKB330RW, independentemente da aplicação do inseticida. O acamamento de 46,7% encontrado para o genótipo DKB330 sem aplicação de inseticida refletiu negativamente na produtividade deste tratamento. Por outro lado, com a aplicação de inseticida, o acamamento variou de 0,2% a 2,2% entre os genótipos, não influenciando sua produtividade. A proteína Cry3Bb1 foi eficaz na redução dos danos na raiz independentemente do genótipo testado. Genótipos com alto potencial produtivo e com menor massa de raiz podem apresentar maior resposta de produtividade quando protegidos com a toxina ou o inseticida na presença de alta população de larvas. Na safra 2010/2011, avaliaram-se os danos na raiz do milho não Bt em quatro épocas de semeadura em Itaberá (14/10, 3/11, 25/11 e 20/12) e três em Castro, PR (29/9, 21/10 e 11/11). Na safra seguinte, de 2011/2012, avaliaram-se seis épocas em Itaberá (21/9, 18/10, 21/11, 28/12, 30/1 e 20/2) e quatro em Castro (20/9, 10/10, 30/10 e 20/11). Constatou-se que, a partir da segunda época, os danos na raiz atingiram nota superior a 3,0 e permaneceram acima de 4,0 em todas as demais épocas de semeadura em Itaberá, nas duas safras avaliadas. Em Castro, apenas a semeadura de 20/11/2011 atingiu nota superior a 3,0. A utilização da proteção na raiz do milho, pela utilização de inseticida ou toxina, apresentou maior resposta nas semeaduras realizadas a partir do mês de outubro na região de Itaberá.

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